A total of 12 Zucker rats, comprising 6 lean and 6 obese rats, all male and 12 weeks old were used in the present study. The present study opted to use only males to reduce variability associated with hormonal differences, which can markedly influence outcomes in studies related to cardiovascular function, sympathetic nervous system activity and metabolic processes. These rats were obtained from the Center for the Development of Experimental Models for Biology and Medicine (CEDEME) at the Federal University of São Paulo (UNIFESP), São Paulo, Brazil. To prevent and alleviate pain, discomfort and distress, the animals were euthanized following an acclimation period of 15 days. The experiment lasted for 17 days. All individuals responsible for inspecting the animals were trained veterinarians, specialized in evaluating the physiology, behavior and overall condition of the animals. All rats were housed in plastic cages (2 animals/cage) in an experimental room maintained under controlled conditions of temperature (22±2˚C) and humidity (50±10%) with 12-h light-dark cycle and ad libitum access to standard feed and water at the Animal Facility of the Faculty of Veterinary Medicine and Animal Science of the University of São Paulo.

The animals were inspected daily, and no abnormalities or health issues were reported during the experimental period; thus, no interventions were necessary. The rats were euthanized using sodium pentobarbital (Cristália Produtos Químicos e Farmacêuticos Ltdal; 200 mg/kg, intraperitoneal injection). The animals were healthy at the time of euthanasia. Animal death was confirmed by a combination of the cessation of heartbeat and respiration, the loss of corneal and toe-pinch reflexes, paleness of the mucous membranes, or the presence of rigor mortis.

The present study was conducted following the approval of the Ethics Committee for the Use of Animals (CEUA) of the Faculty of Veterinary Medicine and Animal Science at the University of São Paulo (CEUA/FMVZ, protocol no. 7564110418).

All personnel involved in the experiment possessed the knowledge and skills to handle the animals in order to avoid pain and discomfort. The rats were individually weighed (g) using an electronic scale (Mettler Toledo Indústria e Comércio Ltda). Immediately after weighing, blood samples (1 ml) were collected from all the rats through tail vein puncture (11), using a venous catheter attached to a hypodermic syringe (BD Precision Glide, Becton & Dickinson Indústria Cirúrgica Ltda). These blood samples were used for the measurements of triglyceride (mg/dl) and total cholesterol (mg/dl) levels.

Blood aliquots were stored in microtubes without ethylenediaminetetraacetic acid (EDTA), individually labeled and processed at the Clinical Laboratory of the Veterinary Hospital of the University of Franca using conventional techniques (12). The samples were processed in an automated biochemical analyzer (Chemwell model-Labtest Diagnóstica) using the Liquiform Triglycerides kit and Liquiform Cholesterol kit (Labtest Diagnóstica S.A.) for triglyceride and total cholesterol measurements, respectively, both based on the enzymatic-Trinder methodology (13).

Following blood collection, the rats were euthanized using sodium pentobarbital (Cristália Produtos Químicos e Farmacêuticos Ltda; 200 mg/kg, intraperitoneal injection) (14).

Subsequently, the thoracic cavities were accessed using a conventional technique (15), to cannulate the ascending aorta for perfusion with phosphate-buffered saline (PBS; Laborclin^®), followed by 4% paraformaldehyde (PFA) (Neon Reagentes Analíticos) in PBS to wash the vascular system.

Subsequently, the hearts were removed from the thoracic cavity and weighed (g) using a precision semi-analytical scale (Mettler Toledo Indústria e Comércio Ltda).

The total volumes of the left ventricles were estimated using the Cavalieri method (16-18). For this purpose, the cardiac chambers were sectioned into ~1-mm-thick fragments perpendicular to the base-apex axis and photographed (Canon^® EOS 400D coupled with a digital Sigma lens 1:1,000 macro). A point system was randomly overlaid on each of the photographic images, with points counted that touched both the wall and the lumen of this cardiac compartment (Fig. 1). The total volume of the left ventricle was estimated by summing the wall volume and lumen wall; V(W) + V(L) in cm³. The following formula was used to calculate the volume of the left ventricle:

Volume of the left ventricle: V(LV)=[∑p(w) x a(pw)] + [∑p(L) x a(pl)] xt

where ∑p represents the sum of points touching the left ventricular wall (w) and the lumen of left ventricle (L); a(p) represents the area associated with each point of the point grid area per point (W): 0.043 cm²; and t represents section thickness at 20 µm.

Total volume of cardiac muscle fibers. After estimating the total volume of the left ventricle, the fragments of this cardiac compartment were sectioned into various sizes and organized in ascending order following the smooth fractionator principle (16,17) (Fig. 1). Subsequently, these fragments were fixed in a 4% formaldehyde solution (Neon Reagentes Analíticos) for 72 h at a temperature of 4˚C and then in a cryoprotective solution (30% sucrose, Neon Reagentes Analíticos) for 24 h at a temperature of 4 to 8˚C. They were embedded in Tissue Tek (Tissue-Tek O.C.T. Compound, Sakura) and frozen in liquid nitrogen at -180˚C. The blocks were cut to a thickness of 20 µm using a cryostat (CM1850; Leica Biosystems, Inc.) and the slides were stained with toluidine blue (MilliporeSigma), at room temperature for 2 min, for observation under a light microscope (Nikon Eclipse 80i; Nikon Corporation) at the Advanced Imaging Diagnostics Center of the School of Veterinary Medicine and Animal Science at the University of São Paulo (CADI-FMVZ USP). The sections were sampled following the SURS pattern and over each histological section, a point-counting system was overlaid (Fig. 2). The points touching the muscle fibers (Vv FM) were counted. The ratio between the number of points hitting the muscle fiber (FM) and the entire myocardium (M) was estimated as follows:

Vv (FM)=Σp (FM)/Σp (M)

To estimate the total volume of muscle fibers (VFM) in mm³, the following formulas were applied:

V(FM)=Vv (FM) xV(W)

where V(FM) represents the total volume of muscle fibers (in mm³), Vv (FM) represents the volume fraction of muscle fibers and V(W) represents the total volume of the left ventricle wall estimated previously.

Total volume of neurons in the stellate ganglia. To generate uniform and random vertical sections, the right and left stellate ganglia (Fig. 3) were rotated along their major axis and subsequently processed for cryostat sections (CM1850; Leica Biosystems, Inc.). The sections were sampled following the SURS pattern and a point system was overlaid on each histological section.

To estimate the total volume of neurons in the stellate ganglia, the Vv (neuron) was multiplied by the total volume of the stellate ganglia (Vge), previously calculated using the Cavalieri's Principle. For the calculation of Vge, a random point system was overlaid on the histological sections of the stellate ganglia:

V(ge)=∑p [a(p) x t x k]

where ∑p represents the summation of points that touched the stellate ganglia; a(p) represents the area associated with each point of the point grid=330 µm²; t represents the thickness of the section=15 µm; and k represents the distance=5 sections.

For the estimation of the total volume of neurons in the stellate ganglia, the following formula was applied:

V(NGe)=Vv (neuron) xVge.

Vv was estimated as follows:

Vv (Neuron)=∑p (N)/∑p (G)

where ∑p (N) represents the summation of points that intersected the neurons and ∑p (G) represents the summation of points that intersected the stellate ganglia.

The frequency distribution of the estimated parameters was conducted using the Shapiro-Wilk test. Non-parametric data were compared using the Mann-Whitney U test and parametric data were compared using the unpaired Student's t-test. Correlation analyses were performed using Spearman's correlation analysis. These analyses were performed using the statistical software GraphPad Prism 8.4.3 (Dotmatics). The results are expressed as the group median followed by the coefficient of variation in parentheses. A P-value <0.05 was considered to indicate a statistically significant difference.

The body weight of the rats was 279.58 g (0.01) and 452.12 g (0.09) for the Zucker lean and fat rats, respectively. When comparing the body weights of the Zucker lean and Zucker fat rats, the difference between groups was significant (P<0.05; Fig. 4A).

The cholesterol level for the Zucker lean rats was 75.6 mg/dl (0.06) and 145.16 mg/dl (0.01) for the Zucker fat rats. The triglyceride level for the Zucker lean rats was 30.36 mg/dl (0.41) and 504.16 mg/dl (0.42) for the fat rats. For both cholesterol and triglyceride measurements, the differences between groups were statistically significant (P<0.05; Fig. 4B and C).

The ventricular lumen volume was 0.36 cm³ (0.32) and 0.75 cm³ (0.18) for the Zucker lean and fat rats, respectively. The total volume of ventricular lumen differed significantly when comparing the groups (P<0.05; Figs. 5A, 6A and B).

Total volume of ventricular wall. The ventricular wall volume was 0.67 cm³ (0.3) and 0.88 cm³ (0.17) or the Zucker lean and Zucker fat rats, respectively. When comparing the groups, the total volume of the ventricular wall did not differ significantly between the groups (P>0.05; Figs. 5B, 6A and B).

Total volume of left ventricle. The left ventricle volume was 1.24 cm³ (0.27) and 1.60 cm³ (0.12) for the Zucker lean and Zucker fat rats, respectively. When comparing the groups, the total volume of left ventricle differed significantly (P<0.05; Fig. 5C).

Total volume of cardiac muscular fiber. The cardiac muscular fiber was 1.16 mm³ (0.09) for the Zucker lean rats and 2.34 mm³ (0.20) for the Zucker fat rats. The total volume of cardiac muscular fiber differed significantly between the groups (P<0.05; Figs. 5D and 7).

Total volume of neurons in stellate ganglia. The volume of neurons in stellate ganglia was 34.29x10⁶ µm³ (0.20) and 23.01x10⁶ µm³ (0.16). The total volume of neurons in the stellate ganglia differed significantly between the Zucker lean and Zucker fat rats (P<0.05; Fig. 5E).

Correlation analysis. A correlation analysis was performed between the following parameters: body weight, left ventricle volume, and neuron volume in the stellate ganglia (Fig. 7).